| ObjectiveTo discuss the effects of acupuncture on depression behavior and nerve function defect degree in hypoxic-ischemic post stroke depression(PSD)rats,to observe the influences of acupuncture on brain morphological changes in hypoxic-ischemic PSD rats,to evaluate the effect of acupuncture on movement disorder and cognitive affective disorder caused by hypoxic-ischemic brain damage.Starting from the theory of "neuroinflammatory response",this paper discussed the role of neuron inflammatory necrosis in the occurrence and development of PSD,discussed the effect of acupuncture on the neuron autophagy in PSD rats,and elucidated the mechanism of acupuncture mediated by regulating neuron autophagy inhibit the neuron inflammatory necrosis in PSD rats.The theory that "autophagy degradation may slow down neuroinflammatory response"reveals the mechanism of acupuncture in improving movements dysfunction and depressive symptoms in PSD rats.MethodsShort-term middle cerebral artery occlusion(MCAO)was used to es t ablish a right cerebrol artery embolization reperfusion modol(temporary middle cerebral artery occlusion/reperfusion,tMCAO/R),lasting for four days after surgery,a post Stroke depression(PSD)model was established with a combination of chronic loneliness and chronic unpredictable mild stress(CUMS),only two stimuli per day(10 in total,two stimuli per day per random-numbered chart),for 21 consecutive days.Give PSD rats acupuncture intervention(GB13 and GV24)and drugs(paroxetine hydrochloride tablets,according to the dose of 10 mg/kg lavage dosing)intervention,intervention once a day,intervention in 14 days in a row,in the process of intervention consistently give CUMS stimulation.All the rats were observed respectively in MCAO made die before,CUMS model,7 acupuncture intervention and acupuncture intervention in 14 days after detection.The experiment was divided into four parts:(1)to evaluate the effects of acupuncture on the behavior of PSD rats(open-field experiment,grooming frequency and sucrose preference rate)and the degree of neurological impairment;(2)TUNEL and TTC detection techniques were used to observe the effects of acupuncture on ischemic and hypoxic brain injury and apoptosis of neurons(hippocampal neurons and prefrontal cortex neurons)in PSD rats;(3)Using Western Blot technique to observe the influence of acupuncture on the expression of autophagy related proteins(Beclin-1,LC3II/I,p62,Bcl-2)in the hippocampal neurons and the prefrontal cortex neurons of the PSD rats,immunofluorescence technique to observe the effect of acupuncture on LC3 positive expression,the influence of the amount of transmission electron microscopy techniques to observe PSD rat hippocampus and the prefrontal cortex neuron ultrastructure and autophagosome morphological changes,HE staining was used to observe the effect of acupuncture on the pathological morphology of cerebral tissue in PSD rats,and the mechanism of acupuncture regulating the autophagy level of neurons in PSD rats to improve the neurological function defect and affective disorder was preliminarily discussed;(4)Using western blot technique to observe the influence of acupuncture on the expression of NF-? B,NLRP3,IL-1? and IL-18,immunofluorescence technique to observe the effect of acupuncture on the positive expression of NLRP3,ASC,Caspase-1 in the hippocampus of the PSD rats,transmission electron microscopy techniques to observe PSD rat hippocampus and the prefrontal cortex neurons inflammatory corpuscle and autophagosome ultrastructure and morphological changes,evaluation of autophagy and inflammatory necrosis of neurons in acupuncture to improve neurologic deficits caused by ischemia hypoxia brain injury and the role of depressive symptoms,clarify acupuncture regulate autophagy:inhibition mechanism of PSD rat inflammatory necrosis of neurons.Results(1)The first part of the experimental study evaluated the success of the cerebral injury model of ischemic stroke prepared by MCAO thread suppository method,and evaluated the success of the depression model of ischemic stroke prepared by orphan combined with CUMS stimulation method by open field experiment,number of grooming and sucrose preference experiment.According to the number of kuang field experiment,grooming(also called modified movement)and sucrose preference experimental results,we found that compared with normal group,PSD model group rats level activities,vertical activity times,number of grooming and sucrose preference rate significantly decreased(P<0.001),suggesting the rats after building appear obvious behavior and symptoms of depression,and acupuncture group the levels of PSD rat activity times,number of vertical activities,grooming times and sucrose preference rate of rats in the model group increased(P<0.001),We believe that acupuncture can significantly improve the symptoms of anhedonia and decreased mobility in depression after ischemic stroke.Symptoms of decreased activity and interest were also reduced in the paroxetine group.Rats compared with normal group,control group,the degree of nerve function defect score has no obvious change,there was no statistically significant difference(P>0.05),PSD model group rats significantly increased degree of nerve function defect score,the difference was statistically significant(P<0.001),suggesting control is not a substitute for MCAO ischemia reperfusion rat neural function damage caused by surgery,PSD model rats significantly impaired nerve function,showing a certain degree of limb movement disorders,such as walking in circles the left or to the left,left fore twin acute inextensible left limbs of hemiplegia symptoms,drooping eyelids obviously on the right side;Compared with PSD rat model group,sham acupuncture group degree of nerve function defect score has no obvious change,there was no statistically significant difference(P>0.05),the degree of nerve function defect score acupuncture group rats decreased significantly,the difference was statistically significant(P<0.001),paroxetine drug group rats nerve function defect rate is reduced,the difference was statistically significant(P<0.05),shows that acupuncture can improve the PSD rat nerve function defect,but paroxetine for PSD rat not satisfied with the effect of nerve dysfunction.(2)The second part of the experimental study found that compared with the control rats,the rat MCAO group of brain ischemia area increased significantly,the difference was statistically significant(P<0.01),PSD model group rats ischemic area increased significantly,the difference was statistically significant(P<0.01),shows that the temporary right middle cerebral artery ischemia reperfusion model experiment preparation,and PSD model rats ischemic area is more serious,may be related to emotional disorders after stroke and the interaction between stroke;Compared with PSD rat model group,sham acupuncture group of brain ischemia area has no obvious change,there was no statistically significant difference(P>0.05),the brain ischemia area of acupuncture group was obviously lower,the difference was statistically significant(P<0.05),paroxetine drug group of ischemia rats area reducing,but there was no statistically significant difference(P>0.05),shows that acupuncture can effectively reduce PSD rat ischemic area,reducing the level of brain infarction is helpful for restoration of nerve function,and paroxetine drug no significant to the improvement of the degree of ischemic brain injury.In the MCAO model group and the PSD model group,the hippocampaus and cortex neurons showed obviously apoptosis(P<0.001).Acupuncture group of PSD rat hippocampal neurons and the number of cortical neurons apoptosis and phenomenon significantly reduced(P<0.001),suggesting acupuncture improving PSD neurologic deficits and emotional disorders may be associated with inhibiting neuronal apoptosis and the mechanism of paroxetine can effectively reduce PSD rat hippocampal neurons and apoptosis of cortical neurons(P<0.001),paroxetine for modern drugs,to some extent can improve depressive symptoms in the treatment of PSD body,inhibit neuronal apoptosis is one of the possible mechanisms of antidepressant.(3)In the third part of the experimental study,we observed the changes of motor function,activity interest,depressive symptoms and neurological impairment score of PSD rats in each group at four time points,including before MCAO modeling,after CUMS modeling(before intervention),on the 7th day and the 14th day of intervention.We found that acupuncture and paroxetine can effectively improve the PSD rat novel psychology,ability to adapt to new things and depressive symptoms,acupuncture can improve the ischemia hypoxia brain injury,repair defect nerve function,promote the recovery of PSD rat limb activity,and internal environment and paroxetine in improving ischemia repair did not show the advantage of nervous functions,3-MA group of motor function,activities of PSD rat interest,depressive symptoms and nervous function defect score significantly in the model group,compared with group 3-MA,The degree of neurological impairment and depressive symptoms of the rats in the acupuncture+3-MA group were significantly reduced,suggesting that acupuncture may have a regulatory effect on autophagy of neurons in PSD rats,and it may improve neurological impairment and cognitive and emotional disorder caused by ischemic and hypoxic brain injury by promoting autophagy flow.We found that beclin-1,BNIP3,and LC3?/? expression were significantly increased(P<0.001,P<0.001,P<0.01),bcl-2 expression was decreased(P<0.001),beclin-1,BNIP3,and LC3?/? expression were significantly increased(P<0.001,P<0.001,P<0.05),and bcl-2 expression was decreased(P<0.001,P<0.001,P<0.05).The LC3 positive expression in the hippocampal area of PSD rats was observed by immunofluorescence technique,and the morphology of neurons in the model group was observed under transmission electron microscopy,with an irregular shape,an indention in one place,an enlarged perinuclear space,a swollen mitochondria,and scattered autophagy bodies.Acupuncture significantly increased the expressions of beclin-l,BNIP3,LC3II/I,and bcl-2 in the hippocampal and prefrontal cortex tissues of rats(P<0.001,P<0.001,P<0.001,P<0.001,P<0.001),and the positive expressions of LC3 in the hippocampus of rats were increased,improving the ultrastructure of neurons and oxidative stress injury.In the paroxetine group,beclin-1,BNIP3,LC3II/I and bcl-2 expression were increased in the hippocampus(P<0.01,P<0.001,P<0.05,P<0.001),and beclin-1,BNIP3,LC3?/? and bcl-2 expression were increased in the prefrontal cortex(P<0.001,P<0.001,P<0.05,P<0.001).LC3 positive expression was increased in the hippocampus of the rats,and the nuclear membrane of neurons was less smooth and there were fewer organelles.In the sham acupuncture group,beclin-1,BNIP3,LC3?/? and bcl-2 expressions in the hippocampus and prefrontal cortex showed no changes(P>0.05,P>0.05,P>0.05,P>0.05).3-MA group of rat hippocampus and cortex of frontal lobe Beclin 1,BNIP3,I.C3II/I,the Bcl-2 expression significantly decreased(P<0.001,P<0.001,P<0.001,P<0.001),the hippocampus of rats reduced JC3 positive expression,autophagy level significantly inhibited neurons,and accompanied by neuron degeneration,swollen mi t ochondria,rough endoplasmic rct iculum expansion and visible "fragmented" inflammatory corpusele cells scattered around the swelling of mitochondria,in neurons in PSD body autophagy and apoptosis may present negative correlation;Compared with 3-MA group,acupuncture+3-MA group in the rat hippocampus and cortex of frontal lobe Beclin 1,BNIP3,LC3?/?,the Bcl-2 expression significantly higher(P<0.001,P<0.001,P<0.001,P<0.001),the rat hippocampus LC3 positive expression enhancement,ischemic anoxia dramatically improve pathological state,cytolysosome and organelles increased significantly,did not see obvious inflammatory corpuscle,rough endoplasmic reticulum expansion phenomonon caused by the ischemic hypoxia was significantly reduced,neurons form trend is good.The results of HE staining were consistent with the above results.(4)In the fourth part of the experimental study,the neurons in the prefrontal cortex of the sham operation group had good morphology and abundant organelles.Compared with control group,PSD model group the hippocampus and prefrontal cortex areas,the NF-?B,NLRP3,IL-1?,IL-18 expression increased(P<0.001,P<0.001,P<0.001,P<0.001),immunofluorescence result in PSD rat on the right side of the brain hippocampus NLRP3,ASC-1,Caspase-1 positive expression is significantly enhanced,hippocampus and cortex neuron morphological variation,rough endoplasmic reticulum expansion,cytolysosome,mitochondria swelling,golgi vacuoles,It suggests that depression brain injury after ischemic stroke can cause the increase of inflammatory level,which proves that autophagy activation after ischemic brain injury in PSD body starts the process of apoptosis process turning to inflammatory necrosis without timely intervention.Compared with PSD model group,acupuncture group on the right side of the brain of rats hippocampus and cortex of frontal NF-?B,NLRP3,IL-1?,IL-18 expression decreased significantly(P<0.001,P<0.001,P<0.001,P<0.001),nuclear membrane of neurons is smooth,rich organelles,cytolysosome significantly increased,acupuncture can significantly improve the PSD the organic brain hippocampus neuron inflammatory response.The expressions of NF-?B,NLRP3,IL-1?,IL-18 in the hippocampus of the paroxetinegroup were increased(P<0.001,P<0.001,P<0.001,P<0.001),and the expressions of NF-?B,NLRP3,IL-1?,IL-18 in the prefrontal cortex were significantly decreased(P<0.001,P<0.01,P<0.01,P<0.001).Immunofluorescence results showed that the positive expressions of NLRP3,ASC-1 and Caspase-1 were decreased in the right hippocampus of the acupuncture group and the paroxetine group.This suggests that the mechanism of paroxetine in improving the mood disorder and anti-depression in PSD may be related to improving the inflammatory response of damaged neurons,but it has no significant effect on improving the ultrastructure of neurons.Compared with PSD model group,sham acupuncture group of hippocampus and prefrontal cortex areas,the NF-?B,NLRP3,IL-1?,IL-18 express no change(P>0.05,P>0.05,P>0.05,P>0.05),sham acupuncture group NLRP3,ASC-1,Caspase-1 positive expression has no obvious change,vacuoles degeneration of neurons,there are several autophagosome,inflammatory cell necrosis broken pieces produced by the material clear and easy to see,mitochondrial cristae off vacuoles degeneration.The expressions of n NF-?B,NLRP3,IL-1?,IL-18 in the hippocampus of the 3-MA group were significantly increased(P<0.001,P<0.001,P<0.001,P<0.001),and the expressions of NF-?B,NLRP3,IL-1?,IL-18 in the prefrontal cortex were significantly increased(P<0.001,P<0.001,P<0.01,P<0.001).NLRP3,ASC-1,Caspase-1 positive expression enhanced obviously,nuelear membrane of neurons roughly,organelles cavitation,nuclear weeks gap widened,cytolysosome not seen,mitochondria organelles surrounding fragmented inflammatory substances is clearly visible,that inhibit autophagy,damaged neurons inflammation is aggravating,to accelerate the apoptosis of the cells from inflammatory necrosis change,so that the damaged neurons function further deterioration;Compared with the 3-MA group,the expressions of NF-?B,NLRP3,IL-1?,IL-18 in the hippocampus of the acupuncture+3-MA group were significantly increased(P<0.001,P<0.001,P<0.001,P<0.001),the expressions of NF-?B,NLRP3,IL-1?,IL-18 in the prefrontal cortex were significantly decreased(P<0.001,P<0.001,P<0.01,P<0.001),the positive expressions of NLRP3,ASC-1,and Caspase-1 were significantly decreased,and the nuclear membranes of neurons were continuous.The Nucleolus is clearly visible,cytolysosome,content rare cells burst,cytolysosome significantly increased,occasional inflammatory necrosis of neurons phenomenon,confirmed that the acupuncture can autophagy level,by raising neurons inhibit cell inflammatory damage,reduce the inflammatory reaction of ischemic brain tissue PSD,improve the PSD of nerve function defect and depressive symptoms.ConclusionThe neuronal inflammatory necroksi s induced by neuroinflammatory roeaction is related to the neural function defect,cognitive affective disorder and abnormal emotion mechanism in PSD rats.Paroxetine can inhibit apoptosis of neurons to improve PSD rat depressive symptoms,and depressive symptoms relief may help reduce the burden of nerve function,but to improve the nature of ischemia hypoxia brain injury had obvious therapeutic effect,suggest the pathogenesis of PSD and the pathogenesis of depression are fundamentally different.Acupuncture can effectively improve the ultrastructure of neurons in PSD rats,and the limb movement disorder and emotional abnormality caused by nerve function defect are related to the activation of neuronal autophagy flow.Acupuncture by promoting autophagy remove damaged neurons,or save the neurons that autophagy as a neuronal protection factors inhibiting the inflammatory responses of neurons,to protect neurons from inflammatory infiltrates injury,improve blood anoxic environment,promotion of nerve function defect repair and improvement after ischemic stroke depressive symptoms,reveals the acupuncture improving PSD rat limb movement disorders and the mechanism of action of depressive symptoms. |
PDF Full Text Request